Molecular Mechanisms Underlying Tak1 Function in Osteoclasts

破骨细胞中 Tak1 功能的分子机制

• 批准号: 8830431
• 负责人: YOUSEF ABU-AMER
• 金额: $ 32.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-07 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8830431
• 关键词: Ablation; Adaptor Signaling Protein; Address; Albers-Schonberg disease; Area; Binding; Biology; Bone Development; Bone remodeling; Cells; Complex; Coupled; Development; Dominant-Negative Mutation; Genes; Genetic; Growth; Health; Homeostasis; In Vitro; Inflammatory; Knock-in Mouse; Knockout Mice; Link; Lysine; MAPK14 gene; MAPK8 gene; Macrophage Colony-Stimulating Factor; Maintenance; Marrow; Mediating; Mitogen-Activated Protein Kinases; Molecular; Mus; Myelogenous; NOTCH1 gene; Neurons; Null Lymphocytes; Osteoblasts; Osteoclasts; Osteolysis; PTB Domain; Pathology; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Phosphotyrosine; Post-Translational Protein Processing; Proteins; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Skeletal Development; Stream; TNFSF11 gene; TRAF6 gene; TRANCE protein; Testing; Thick; Tooth eruption; Transcriptional Regulation; Transforming Growth Factor beta; Ubiquitination; base; bone; bone loss; cell type; microCT; notch protein; novel; osteoclastogenesis; protein degradation; receptor; response; restoration; substantia spongiosa; transcription factor; ubiquitin-protein ligase; upstream kinase

DESCRIPTION (provided by applicant): Bone development is tightly regulated by bone forming cells, osteoblasts, and bone resorbing cells, osteoclasts. Therefore, understanding the mechanisms governing osteoclastogenesis is crucial for addressing bone loss pathologies. Differentiation of osteoclasts is governed by RANK ligand which activates several signal transduction pathways, including MAP kinases and NF-κB pathways. Proximal activation entails recruitment of TRAF6 and other key proteins including TGF-ß-activated kinase-1 (TAK1) to the receptor RANK. TRAF6, TAK1 and other signaling partners undergo extensive post-translational modifications aimed at stabilizing RANK signaling and enabling precise regulation and execution of proper down stream signals, primarily NF-κB activation. Precise regulation of NF-κB activity is crucial to maintain normal osteoclast activity and bone homeostasis. Conversely, abnormal activity of this transcription factor causes deleterious inflammatory osteolysis. In fact, we discovered recently that constitutive activation of IKK2 is sufficient to induce RANKL-independent osteoclastogenesis in vitro. More convincingly, we reported that knock-in of constitutively active IKK2 causes severe bone loss in mice. Given that IKK2 phosphorylation and activation is governed by TAK1, a MAP kinase heavily implicated in poly-ubiquitination and stabilization of RANK-TRAF6 complexes and down-stream signaling, we decided to investigate its molecular role in osteoclastogenesis. Thus, we generated mice harboring myeloid-specific deletion of TAK1. These mice displayed all hallmarks of osteopetrosis primarily defective osteoclastogenesis. Mechanistically, we observed that Tak1-null precursors fail to generate osteoclasts. More importantly, we discovered diminished expression of key osteoclastogenic proteins including TRAF6, NEMO and Notch-NICD. This phenomenon was associated with accumulation of NUMBL, a previously described neuron protein. Consistent with these observations, we established that exogenous expression of NUMBL induces degradation of TRAF6, NEMO, NOTCH1-NICD, and inhibits osteoclastogenesis in vitro. Inhibition of NUMBL using a dominant negative PTB-phosphotyrosine-binding of NUMBL and shRNAs knockdown of NUMBL enhanced expression of TRAF6 and NEMO and did not inhibit osteoclastogenesis in wild-type cells. In addition, inhibition of NUMBL using a dominant negative PTB of NUMBL and exogenous expression of NOTCH1-NICD restored osteoclastogenesis in TAK1-null cells. Based on these observations we hypothesize that "NUMBL is a repressor of osteoclastogenesis and its expression is regulated by TAK1. Deletion of TAK1 leads to accumulation of NUMBL protein which induces degradation of TRAF6, NEMO and NICD proteins, and subsequently blocks osteoclastogenesis." To test this hypothesis, we propose to investigate the following specific aims: 1) Determine the mechanism by which TAK1 regulates NUMBL expression. 2) Determine the mechanism by which TAK1 deletion regulates and impedes expression of TRAF6, NEMO and NICD. 3) Determine the effect of genetic ablation of NUMBL on the osteopetrotic phenotype of TAK1-null mice.

描述（由申请人提供）：骨发育受骨形成细胞、成骨细胞和骨吸收细胞、破骨细胞的严格调节。因此，了解破骨细胞生成的机制对于解决骨丢失病理学至关重要。破骨细胞的分化由RANK配体控制，RANK配体激活几种信号转导途径，包括MAP激酶和NF-κB途径。近端激活需要TRAF 6和其他关键蛋白包括TGF-β激活的激酶-1（TAK 1）募集到受体RANK。TRAF 6、TAK 1和其他信号传导伴侣经历了广泛的翻译后修饰，旨在稳定RANK信号传导，并能够精确调节和执行适当的下游信号，主要是NF-κB激活。NF-κB活性的精确调节对于维持正常的破骨细胞活性和骨稳态是至关重要的。相反，这种转录因子的异常活性导致有害的炎性骨质溶解。事实上，我们最近发现IKK 2的组成性激活足以在体外诱导RANKL非依赖性破骨细胞生成。更令人信服的是，我们报告说，敲入组成型活性IKK 2导致严重的骨丢失的小鼠。鉴于IKK 2磷酸化和激活受TAK 1（一种与多聚泛素化和RANK-TRAF 6复合物稳定化以及下游信号传导密切相关的MAP激酶）控制，我们决定研究其在破骨细胞生成中的分子作用。因此，我们产生了TAK 1骨髓特异性缺失的小鼠。这些小鼠表现出所有的特征，主要是破骨细胞生成缺陷。从机制上讲，我们观察到Tak 1-null前体不能产生破骨细胞。更重要的是，我们发现关键的破骨细胞生成蛋白包括TRAF 6、NEMO和Notch-NICD的表达减少。这种现象与NUMBL（一种先前描述的神经元蛋白）的积累有关。与这些观察结果相一致，我们建立了NUMBL的外源性表达诱导TRAF 6，NEMO，NOTCH 1-NICD的降解，并抑制体外破骨细胞生成。使用NUMBL的显性负PTB-磷酸酪氨酸结合和NUMBL的shRNA敲低来抑制NUMBL增强TRAF 6和NEMO的表达，并且不抑制野生型细胞中的破骨细胞生成。此外，使用NUMBL的显性负性PTB和NOTCH 1-NICD的外源性表达抑制NUMBL恢复了TAK 1-null细胞中的破骨细胞生成。基于这些观察，我们假设“NUMBL是破骨细胞生成的阻遏物，其表达受TAK 1调节。TAK 1的缺失导致NUMBL蛋白的积累，NUMBL蛋白诱导TRAF 6、NEMO和NICD蛋白的降解，并随后阻断破骨细胞生成。为了验证这一假设，我们提出了以下具体目标：1）确定TAK 1调节NUMBL表达的机制。2)确定TAK 1缺失调控和阻碍TRAF 6、NEMO和NICD表达的机制。3)确定NUMBL基因消除对TAK 1缺失小鼠的骨硬化表型的影响。